1. Field of the Invention
The present invention relates to a method of isolating phenolic secondary plant constituents from plant material, and to a food additive obtainable by this method.
2. Description of the Related Art
Plant constituents, in particular what are known as the secondary plant constituents, which have no substantial calorific value, have increasingly attracted the attention of science and food technology, where they are of interest as food supplements or food additives by the key phrase “functional food”.
The compounds which are of particular importance in this context are low-molecular-weight compounds which have antioxidative properties and, as a consequence, are capable of inactivating the ubiquitously generated so-called “reactive oxygen species”, that is reactive oxygen-comprising molecules such as, for example, hydroxyl radicals OH., the superoxide anion O2−, hydrogen peroxide H2O2, singlet oxygen which is capable of reacting with nitrogen monoxide NO to give peroxynitrite ONO2, and hypobromite and hypochlorite. Further positive effects of these low-molecular-weight compounds which are discussed intensively in connection with their antioxidative activity are, for example, the protection of endothelial cells, the suppression of tumor growth and the protection of the cardiovascular system. The cardioprotective activity, for example, which is associated with the regular consumption of red wine, is caused, inter alia, by diphenols which are substituted by OH groups, such as, for example, resveratrol. Resveratrol is a substance which, in turn, probably acts as an antioxidant and lowers the oxidation sensitivity of low-density lipoprotein (LDL) in the blood, inhibits platelet aggregation and inhibits the endogenous cholesterol biosynthesis by inhibiting the squalene monooxygenase enzyme.
The technical isolation, or obtaining, secondary plant constituents makes particularly high demands on the work-up thereof since the stability of many isolated substances declines rapidly above a certain pH. Since in addition many plant extracts have a high fruit acid and ion content, separation via traditional ion-exchange chromatography is made difficult or indeed impossible. Furthermore, methods known from the prior art frequently require large volumes of plant extract to be employed, require toxicologically unacceptable, or caustic, substances such as methanol or acetic acid for eluting the secondary plant constituents, and are frequently very time-consuming. In contrast, the limited storage stability of many secondary plant constituents requires rapid process steps so as to be able to transfer the sensitive substances as quickly as possible into a stabilizing medium.
An example which is known from the prior art is a method of concentrating anthocyanin from blueberry juice concentrate by means of a strongly hydrophobic Amberlite® XAD column with a length of two meters and a diameter of 11 cm. The disadvantages of this method, however, are the required high volumes of juice concentrate to be employed, the elution with toxicologically unacceptable, or caustic, substances such as methanol and acetic acid, and the high expenditure of time for separating the anthocyanins from the juice concentrate.
WO 2008/136741 A1 discloses a method for removing polyphenols from beverages, in which the beverages are treated with a polymer matrix to which ether ligands, preferably polyether ligands with multiple CC bonds, are fixed. The polymer matrix may be present as a particulate or membrane-shaped adsorbent.
WO 2008/097154 A1 discloses a method for removing turbidity-causing materials from beverages. The polymer matrices which are used are based on crosslinked polysaccharides and are provided with a polyether coating which can be generated by grafting for example polyethylene glycol or diethylene glycol vinyl ether. Providing the matrices with polyether functions makes possible the efficient removal of undesired polyphenols from beverages, where the polyphenols do not constitute target products of the method.
U.S. Pat. No. 5,141,611 discloses a method of removing polyphenols from beverages, where polyamide membranes or polyamide particles with a surface modification based on glutaraldehyde/resorcinol or based on glutaraldehyde in combination with melamine, 1,6-hexamethylenediamine or various amino acids, are used.
EP 0 806 474 A1 discloses a method in which Sepharose-based chromatography gels which have cation-exchanging ligands (sulfopropyl or carboxymethyl groups) are used for removing turbidity from beverages or for stabilizing beverages. In this method, polyphenols together with companion proteins, being undesired contaminants, are removed from beer. It is furthermore disclosed how the cation exchangers can be regenerated for reuse by the action of water, sodium hydroxide solution or saline. The method disclosed in EP 0 806 474 A1 does not contain any steps which permit the isolation of the polyphenols as target substances, i.e. steps which permit the removal of the polyphenols from the accompanying proteins or other contaminants from beer production.
U.S. Pat. No. 5,886,155 discloses a method for the adsorptive removal of tannins and polyphenols from protein mixtures of vegetable origin by means of hydrophobic interaction chromatography (HIC), with the target proteins being eluted from the HIC matrix, and the subsequent purification of the target proteins by means of a second step of hydrophobic interaction chromatography.
WO 00/45769 A2 discloses a method for isolating polyphenolic antioxidants from a purin-comprising plant extract, where the antioxidants are selectively adsorbed to a matrix which is composed of polyvinylpolypyrrolidone, chitosan or mixtures of these, and where the matrix is present for example as a chromatography resin.
In Journal of Membrane Science 134 (1997), 191-197, Z. Borneman et al. disclose a method for removing polyphenols, and of a brown coloration associated with the presence of these polyphenols, from apple juice. As regards the removal of these polyphenols, polyether sulfone membranes (PES) which are modified with polyvinylpyrrolidone outperform membranes of regenerated cellulose which has no ligands fixed to it. Fouling phenomena which are observed on the PES membrane during the method can be reversed by regenerating the membrane with sodium hydroxide solution.